Arrayed mutant haploid embryonic stem cell libraries facilitate phenotype-driven genetic screens

Liu G^1,2, , Wang X^1,2,  Liu Y^1,2,  Zhang M^1,2,  Cai T³, Shen Z³, Jia Y^1,2,  Huang Y^1,2
Nucleic Acids Res. 2017 Sep 28. [Epub ahead of print]
PMID: 29036617, DOI: 10.1093/nar/gkx857
 
Abstract
Forward genetic screens using mammalian embryonic stem (ES) cells have identified genes required for numerous cellular processes. However, loss-off-function screens are more difficult to conduct in diploid cells because, in most cases, both alleles of a gene must be mutated to exhibit a phenotype. Recently, mammalian haploid ES cell lines were successfully established and applied to several recessive genetic screens. However, all these screens were performed in mixed pools of mutant cells and were mainly based on positive selection. In general, negative screening is not easy to apply to these mixed pools, although quantitative deep sequencing of mutagen insertions can help to identify some ‘missing’ mutants. Moreover, the interplay between different mutant cells in the mixed pools would interfere with the readout of the screens. Here, we developed a method for rapidly generating arrayed haploid mutant libraries in which the proportion of homozygous mutant clones can reach 85%. After screening thousands of individual mutant clones, we identified a number of novel factors required for the onset of differentiation in ES cells. A negative screen was also conducted to discover mutations conferring cells with increased sensitivity to DNA double strand breaks induced by the drug doxorubicin. Both of these screens illustrate the value of this system.

    本所重點(diǎn)實(shí)驗室黃粵課題組的研究論文“Arrayed mutant haploid embryonic stem cell libraries facilitate phenotype-driven genetic screens” 于2017年9月28日在線(xiàn)發(fā)表于《Nucleic Acids Research》。有趣的是，國際著(zhù)名學(xué)術(shù)期刊《Nature》于10月5日也發(fā)表了一篇研究?jì)热菹嗨频难芯空撐摹癆 reversible haploid mouse embryonic stem cell biobank resource for functional genomics”，來(lái)自?shī)W地利Josef Penninger教授研究組。
    利用哺乳動(dòng)物細胞進(jìn)行的遺傳篩選工作已經(jīng)被廣泛的用于揭示基因的生物學(xué)功能，但哺乳動(dòng)物細胞基因組的二倍體性阻礙了基因純合突變體的獲得，最近單倍體胚胎干細胞（Embryonic Stem Cell, ESC）的成功分離很好地解決了這一問(wèn)題。在本研究中，我們利用小鼠單倍體ESC和PiggyBac（PB）轉座子構建了矩陣式突變體文庫，其中85% 以上的克隆是基因純合突變體。我們首先利用該文庫進(jìn)行了正向選擇性篩選，通過(guò)對2024個(gè)突變體克隆進(jìn)行了分化相關(guān)基因的篩選，獲得了40個(gè)與分化缺陷相關(guān)的基因，利用回復實(shí)驗和CRISPR-Cas9技術(shù)在二倍體ESC中突變相關(guān)基因確定了相關(guān)基因和分化缺陷表型間的因果關(guān)系。由于負性選擇性篩選是鑒定丟失的克隆，雖然混合文庫篩選結合二代測序技術(shù)通過(guò)量化篩選前后所有的突變可以鑒定部分丟失的克隆，但混合文庫中不同突變細胞之間的相互影響會(huì )干擾最后篩選的數據，而矩陣式突變體文庫很好的避免了這個(gè)問(wèn)題。在本研究中，我們進(jìn)行了誘導DNA雙鏈斷裂藥物-阿霉素(Doxorubicin)敏感性增加的遺傳篩選，利用高內涵篩選儀從1152個(gè)突變克隆中獲得了13個(gè)藥物敏感性增加的克隆，其中定位到編碼基因的有6個(gè)，回復克隆對阿霉素敏感性得到回復確定了相關(guān)基因與與藥物敏感性之間的關(guān)系。因此，我們建立的矩陣式基因突變文庫擴展了遺傳篩選的應用，具有廣泛的應用前景，并且建立相關(guān)網(wǎng)站為同行提供文庫服務(wù)。
    黃粵課題組的劉光助理研究員、王雪博士（2017年畢業(yè)）和劉語(yǔ)方博士（2015年畢業(yè)）為該研究論文的共同第一作者，其他作者包括黃粵課題組的張美麗助理研究員、賈玉艷助理研究員；北京生命科學(xué)研究所的蔡濤研究員、沈志榮研究員。黃粵研究員為本文通訊作者。該研究工作得到了科技部重點(diǎn)研發(fā)計劃（2016YFA0100103）、中國醫學(xué)科學(xué)院醫學(xué)與健康科技創(chuàng )新工程（2016-I2M-3-002）及國家自然科學(xué)基金（2013CB967002和31671410）等項目的資助。